Transgenic Mitomice Models for Treatment of Blinding Diseases

描述（由申请人提供）：虽然突变的线粒体（mt）DNA与Leber’s遗传性视神经病变（LHON）的失明紧密相关，但具有突变mtDNA复合物I亚基的真正的动物模型尚未开发，该模型将能够探测视神经病变的发病机制并测试潜在的治疗途径。由于将DNA输送到线粒体中存在障碍，因此不可能建立由线粒体突变引起的人类疾病的动物模型。我们通过在腺相关病毒（AAV）的病毒蛋白衣壳上附加线粒体靶向序列（MTS）来绕过这一屏障，从而将突变的人类ND4基因（占所有LHON病例的一半）引导到小鼠线粒体中。当由人类线粒体启动子驱动时，小鼠线粒体中表达突变的人类ND4诱导视力丧失，并伴有视网膜神经节细胞及其组成视神经的轴突的进行性死亡，从而重现人类LHON疾病的特征。我们从事这个项目的动机是为无法治愈的导致失明的线粒体疾病开发基因疗法。为此，我们必须(1)开发一种可行的治疗方法，一种MTS AAV，将正常基因传递到受影响的线粒体；(2)创建具有突变的复合体I亚基基因的动物模型，重现人类疾病，以测试潜在的治疗方法和视神经病变在与人类疾病相似的阶段的发病机制。为了培育转基因小鼠，我们将含有突变的人ND4等位基因的MTS AAV通过显微注射到小鼠囊胚中植入胚胎干细胞，产生的后代在出生一年后具有进行性视网膜神经节细胞（RGC）死亡和视神经病变，视网膜电图（PERG）振幅稳定下降到噪声水平。为了监测活体动物线粒体基因的表达，我们将线粒体编码的红色荧光蛋白（mCherry）添加到含有突变人类ND4的MTS AAV构建物中，该构建物可以通过激光扫描眼镜（LSO）在活体后代及其三代后代中观察到。我们的具体目标从理解小鼠NADH脱氢酶亚基ND4突变体的后果到开发更多的转基因系，表达负责LHON的另外两个突变体人类复合体I亚基（ND1和ND6）。

DESCRIPTION (provided by applicant): While mutated mitochondrial (mt)DNA is firmly linked to the blindness of Leber's hereditary optic neuropathy (LHON), a bona fide animal model with mutated mtDNA complex I subunits that would enable probing the pathogenesis of the optic neuropathy and testing of potential avenues for therapy has yet to be developed. It had not been possible to produce animal models of human diseases caused by mitochondrial mutations due to the barrier in delivering DNA into mitochondria. We circumvented this barrier by appending to the adenoassociated virus (AAV) a mitochondrial targeting sequence (MTS) to the viral protein capsid to direct the mutant human ND4 gene (responsible for half of all LHON cases) into murine mitochondria. When driven by a human mitochondrial promoter, expression of mutant human ND4 in murine mitochondria induced visual loss with a progressive demise of ganglion cells in the retina and their axons comprising the optic nerve, thus recapitulating the hallmarks of the human LHON disorder. Our motivation in pursuing this project is to develop gene therapy for untreatable mitochondrial diseases that lead to blindness. For this, we have to (1) develop a plausible therapy, an MTS AAV to deliver normal genes to affected mitochondria and (2) create animal models with mutated complex I subunit genes that recapitulate the human disorder in which to test potential treatments and the pathogenesis of optic neuropathy at stages that resemble the human disease. To generate a transgenic mouse we delivered the MTS AAV containing the mutant human ND4 allele to embryonic stem cells by microinjection into the blastocyst of the mouse, generating offspring with progressive retinal ganglion cell (RGC) demise and optic neuropathy with pattern electroretinogram (PERG) amplitudes declining steadily to noise levels one year after birth. To monitor mitochondrial gene expression in live animals we added a mitochondrial encoded red fluorescent protein (mCherry) to the MTS AAV construct containing mutant human ND4 that could be visualized by laser scanning ophthalmoscopy (LSO) in live offspring and so far in three generations of their progeny. Our Specific Aims logically progress from understanding the consequences of mutant NADH dehydrogenase subunit ND4 in mice to developing additional transgenic lines expressing the two other mutant human complex I subunits (ND1 and ND6) responsible for LHON.